Voltage-gated sodium (NaV)- and potassium (KV)-channel proteins underlie the regulation of basal electrical excitability and the initiation and repolarization of action potentials in virtually all excitable cells. These channels have evolved from primordial K+-selective pores into diverse proteins with regulatory mechanisms that enable them to respond to specific stimuli in the nervous, cardiovascular, and immune systems. The importance of this protein family to human health is highlighted by the facts that: inherited or acquired defects in NaV or Kv channels cause epilepsy, myotonia, erythromelalgia and cardiac arrhythmias; mutations that lead to changes in the gating kinetics or expression of K+ channels in excitable tissues such as cardiac muscle can lead to arrhythmias and susceptibility to sudden cardiac death (long or short QT syndromes); and significant electrical remodeling of KV- and NaV-channel expression is observed during cardiac hypertrophy or persistent arrhythmias. Unfortunately, a broad spectrum of excitability disorders remains largely untreatable, and a fresh approach to closing the gap in our understanding of NaV and KV gating and selectivity will be needed if effective therapeutics are to be developed. Notably, although ion channels (particularly those of the voltage-gated ion channel superfamily) have been characterized on both the macroscopic and atomic levels, these studies lack the resolution needed to identify essential chemical property(s) of the amino acids that have been implicated in functional roles. The lack of such information remains a significant block to our understanding of ion permeation and channel gating, and ultimately, effective drug design. Here we propose to design and apply powerful synthetic tools, in the form of tailor-made unnatural amino acids, as a means of achieving hypothesis-driven atomic-level mutagenesis to reach a physiological endpoint: an understanding of the basis of ion selectivity and channel gating. Further, although we expect to eventually be able to perform structure-based drug design, no structures yet exist for eukaryotic NaVs, and the work proposed here will inform us directly about which traits of bacterial NaV's (where structures are now available) are relevant to eukaryotic NaV's. Finally, the results of our study will remove a significant technical barrier to an atomic-level, functional understanding of the gating and permeation mechanisms employed by NaV and KV channels-two proven drug targets in the management of excitability disorders. Success of the proposed study will make it possible to generate novel amino acids that will be widely available to the research community.